Evaluation of co-culture of cellulolytic fungi for enhanced cellulase and xylanase activity and saccharification of untreated lignocellulosic material.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Differential Carbon Catabolite Repression and Hemicellulolytic Ability among Pathotypes of Colletotrichum lindemuthianum against Natural Plant Substrates.

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.

The transcription factor STE12 influences growth on several carbon sources and production of dehydroacetic acid (DHAA) in Trichoderma reesei.

The filamentous ascomycete Trichoderma reesei, known for its prolific cellulolytic enzyme production, recently also gained attention for its secondary metabolite synthesis. Both processes are intricately influenced by environmental factors like carbon source availability and light exposure. Here, we explore the role of the transcription factor STE12 in regulating metabolic pathways in T. reesei in terms of gene regulation, carbon source utilization and biosynthesis of secondary metabolites. We show that STE12 is involved in regulating cellulase gene expression and growth on carbon sources associated with iron homeostasis. STE12 impacts gene regulation in a light dependent manner on cellulose with modulation of several CAZyme encoding genes as well as genes involved in secondary metabolism. STE12 selectively influences the biosynthesis of the sorbicillinoid trichodimerol, while not affecting the biosynthesis of bisorbibutenolide, which was recently shown to be regulated by the MAPkinase pathway upstream of STE12 in the signaling cascade. We further report on the biosynthesis of dehydroacetic acid (DHAA) in T. reesei, a compound known for its antimicrobial properties, which is subject to regulation by STE12. We conclude, that STE12 exerts functions beyond development and hence contributes to balance the energy distribution between substrate consumption, reproduction and defense.

Approaches for Producing Fungal Cellulases Through Submerged Fermentation.

Fungal cellulases are the most sought-after biological molecules produced from microbial sources in the last four decades. Owing to their emerging applications in the bioenergy industry for hydrolyzing cellulose, for which they are the most abundant source on this planet, research trends are shifting heavily toward adapting to submerged fermentation. However, filamentous fungal species, which are efficient cellulase producers, are well-adapted to low-moisture solid support as the substrate, such as in nature. Therefore, various fermentation strategies are currently being investigated to adapt them to submerged fermentation for large and high-quality production of cellulases. Emerging research trends, such as the use of inexpensive feedstocks, nutrient and/or culture optimization, innovative bioreactor designs, microparticle-assisted fungal growth, and innovative genetic engineering approaches, are some of the recent efforts by researchers to exploit the full potential of these biological molecules. This review discusses some of these strategies and their success rates in various research conditions. In addition, specific focus was provided to both increasing the market value of cellulases and the innovative strategies required to enhance their production on an industrial scale.

Current perspective in research and industrial applications of microbial cellulases.

The natural interactions between various bacteria, fungi, and other cellulolytic microorganisms destroy lignocellulosic polymers. The efficacy of this process is determined by the combined action of three main enzymes: endoglucanases, exo-glucanases, and ß-glucosidase. The enzyme attacks the polymeric structure's ß-1,4-linkages during the cellulose breakdown reaction. This mechanism is crucial for the environment as it recycles cellulose in the biosphere. However, there are problems with enzymatic cellulose breakdown, including complex cellulase structure, insufficient degradation efficacy, high production costs, and post-translational alterations, many of which are closely related to certain unidentified cellulase properties. These issues impede the practical use of cellulases. A developing area of research is the application of this similar paradigm for industrial objectives. Cellulase enzyme exhibits greater promise in many critical industries, including biofuel manufacture, textile smoothing and finishing, paper and pulp manufacturing, and farming. However, the study on cellulolytic enzymes must move forward in various directions, including increasing the activity of cellulase as well as designing peptides to give biocatalysts their desired attributes. This manuscript includes an overview of current research on different sources of cellulases, their production, and biochemical characterization.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from a Trichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolase II. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

Bioethanol Production from Characterized Pre-treated Sugarcane Trash and Jatropha Agrowastes.

Low production costs and a potential feedstock supply make lignocellulosic ethanol (bioethanol) an important source of advanced biofuels. The physical and chemical preparation of this kind of lignocellulosic feedstock led to a high ethanol yield. In order to increase the yield of fermentable sugars, pretreatment is an essential process step that alters the lignocellulosic structure and improves its accessibility for the expensive hydrolytic enzymes. In this context, the chemical composition of sugarcane trash (dry leaves, green leaves, and tops) and jatropha (shell and seed cake) was determined to be mainly cellulose, hemicellulose, and lignin. Hydrogen peroxide and sodium hydroxide were applied in an attempt to facilitate the solubilization of lignin and hemicelluloses in five agrowastes. The extraction of hydrogen peroxide was much better than that of sodium hydroxide. A comparative study was done using SEM, EDXA, and FTIR to evaluate the difference between the two methods. The pretreated wastes were subjected to saccharification by commercial cellulases (30 IU/g substrate). The obtained glucose was fortified with nutrients and fermented statically by Saccharomyces cerevisiae F-307 for bioethanol production. The results revealed the bioethanol yields were 325.4, 310.8, 282.9, 302.4 and 264.0 mg ethanol/g treated agrowastes from green leaves of sugarcane, jatropha deolied seed cake, tops sugarcane, dry leaves of sugarcane, and jatropha shell, respectively. This study emphasizes the value of lignocellulosic agricultural waste as a resource for the production of biofuels as well as the significance of the extraction process.

Tailoring a cellulolytic enzyme cocktail for efficient hydrolysis of mildly pretreated lignocellulosic biomass.

Commercially available cellulase cocktails frequently demonstrate high efficiency in hydrolyzing easily digestible pretreated biomass, which often lacks hemicellulose and/or lignin fractions. However, the challenge arises with enzymatic hydrolysis of mildly pretreated lignocellulosic biomasses, which contain cellulose, hemicellulose and lignin in high proportions. This study aimed to address this question by evaluating the supplementation of a commercial cellulolytic cocktail with accessory hemicellulases and two additives (H2O2 and Tween® 80). Statistical optimization methods were employed to enhance the release of glucose and xylose from mildly pretreated sugarcane bagasse. The optimized supplement composition resulted in the production of 304 and 124 mg g-1 DM of glucose and xylose, respectively, significantly increasing glucose release by 84% and xylose release by 94% compared to using only the cellulolytic cocktail. This enhancement might be attributed to a coordinated hemicellulases action degrading hemicellulose, creating more space for cellulase activity, potentially boosted by the presence of H2O2 and Tween® 80. However, the addition of different concentrations of H2O2 in combination with hemicellulase and Tween® 80 did not result a significant difference on sugar release, which could be attributed to the limited range of concentrations studied (5 to 65 µM). The results obtained in this study using the mix of three supplements were also compared to the addition of only hemicellulase and only Tween® 80 to the cellulolytic cocktail. A significant increase in glucose release of 39% and 41%, respectively, was observed when using the optimized combination. For xylose, the increase was 38% and 41%, respectively. This study underscores the substantial potential in optimizing enzyme cocktails for the hydrolysis of mildly pretreated lignocellulosic biomass by using enzymes and additive combinations tailored to the specific biomass composition.

ß-glucosidases from Saccharomyces cerevisiae: production, protein precipitation, characterization, and application in the enzymatic hydrolysis of delignified sugarcane bagasse.

ß-glucosidase is an essential enzyme for the enzymatic hydrolysis of lignocellulosic biomass, as it catalyzes the final stage of cellulose breakdown, releasing glucose. This paper aims to produce ß-glucosidase from Saccharomyces cerevisiae and evaluate the enzymatic degradation of delignified sugarcane bagasse. S. cerevisiae was grown in yeast peptone dextrose medium. Partial purification of the enzyme was achieved through precipitating proteins with ethanol, and the optimal activity was measured by optimizing pH and temperature. The effects of ions, glucose tolerance, and heat treatment were evaluated. Delignified sugarcane bagasse was hydrolyzed by the enzyme. ß-glucosidase showed a specific activity of 14.0712 ± 0.0207 U mg-1. Partial purification showed 1.22-fold purification. The optimum pH and temperature were 6.24 and 54 °C, respectively. ß-glucosidase showed tolerance to glucose, with a relative activity of 71.27 ± 0.16%. Thermostability showed a relative activity of 58.84 ± 0.91% at 90 °C. The hydrolysis of delignified sugarcane bagasse showed a conversion rate of 87.97 ± 0.10% in the presence of Zn2+, an ion that promoted the highest increase in enzymatic activity. S. cerevisiae produced an extracellular ß-glucosidase with good stability at pH and temperatures conventionally applied in the hydrolysis of lignocellulosic biomass, showing viability for industrial application.

Enzymatic degradation of cellulose in soil: A review.

Cellulose degradation is a critical process in soil ecosystems, playing a vital role in nutrient cycling and organic matter decomposition. Enzymatic degradation of cellulosic biomass is the most sustainable and green method of producing liquid biofuel. It has gained intensive research interest with future perspective as the majority of terrestrial lignocellulose biomass has a great potential to be used as a source of bioenergy. However, the recalcitrant nature of lignocellulose limits its use as a source of energy. Noteworthy enough, enzymatic conversion of cellulose biomass could be a leading future technology. Fungal enzymes play a central role in cellulose degradation. Our understanding of fungal cellulases has substantially redirected in the past few years with the discovery of a new class of enzymes and Cellulosome. Efforts have been made from time to time to develop an economically viable method of cellulose degradation. This review provides insights into the current state of knowledge regarding cellulose degradation in soil and identifies areas where further research is needed.

Making the biochemical conversion of lignocellulose more robust.

Lignocellulose is an alternative to fossil resources, but its biochemical conversion is not economically competitive. While decentralized processing can reduce logistical cost for this feedstock, sugar platforms need to be developed with energy-saving pretreatment technologies and cost-effective cellulases, and products must be selected correctly. Anaerobic fermentation with less energy consumption and lower contamination risk is preferred, particularly for producing biofuels. Great effort has been devoted to producing cellulosic ethanol, but CO2 released with large quantities during ethanol fermentation must be utilized in situ for credit. Unless titer and yield are improved substantially, butanol cannot be produced as an advanced biofuel. Microbial lipids produced through aerobic fermentation with low yield and intensive energy consumption are not affordable as feedstocks for biodiesel production.

Overexpression of the Transcription Factor Azf1 Reveals Novel Regulatory Functions and Impacts ß-Glucosidase Production in Trichoderma reesei.

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the ß-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these ß-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes.

Different Putative Methyltransferases Have Different Effects on the Expression Patterns of Cellulolytic Genes.

Putative methyltranferase LaeA and LaeA-like proteins, conserved in many filamentous fungi, regulate fungal growth, development, virulence, the biosynthesis of secondary metabolites, and the production of cellulolytic enzymes. Penicillium oxaliucm is a typical fungus that produces cellulolytic enzymes. In this study, we reported the biological function of eight putative methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) containing a methyltransf_23 or methyltransf_25 domain, with a focus on their roles in the production of cellulolytic enzymes. In P. oxalicum, various methyltransferase genes displayed different transcriptional levels. The genes Pomtr23C and Pomtr25A exhibited high transcriptional levels, while Pomtr23D/E/F/G/H and Pomtr25B were transcribed constantly at low levels. The gene deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed. Various mutants have different patterns in cellulolytic enzyme production. Compared to the WT, the largest increase in filter paper activity (FPA, indicating total cellulase activity) was observed in the Δmtr23G mutant, the only mutant with a cellulolytic halo surrounding the colony. Three mutants (Δmtr23C/D and Δmtr25A) also showed increased cellulolytic enzyme production. The Δmtr23E and Δmtr25B mutants displayed decreased FPA activity, while the Δmtr23F and Δmtr23H mutants displayed similar patterns of cellulolytic enzyme production compared with the WT. The assay of transcriptional levels of cellobiohydrolase gene Pocbh1 and ß-1,4-endoglucanase Poeg1 supported that higher cellulolytic gene transcription resulted in higher production of cellulolytic enzymes, and vice versa. The transcriptional levels of two transcription factors, activator XlnR and repressor CreA, were measured. The high transcription level of the PoxlnR gene in the Δmtr23D mutant should be one reason for the increased transcription of its cellulolytic enzyme gene. Both XlnR and CreA transcriptional levels increased in the Δmtr23G mutant, but the former showed a more significant increase than the latter, indicating that the activation effect predominated. The PoMtr25A is localized in the nucleus. The catalytic subunit SNF2 of the SWI/SNF chromatin-remodeling complex was found as one of the interacting proteins of PoMtr25A via tandem affinity purification coupled with mass spectrometry. PoMtr25A may affect not only the transcription of repressor CreA but also by recruiting SWI/SNF complexes that affect chromatin structure, thereby regulating the transcription of target genes.

Effect of the res2 transcription factor gene deletion on protein secretion and stress response in the hyperproducer strain Trichoderma reesei Rut-C30.

BACKGROUND: The fungus Trichoderma reesei is one of the most used industrial cellulase producers due to its high capacity of protein secretion. Strains of T. reesei with enhanced protein secretion capacity, such as Rut-C30, have been obtained after several rounds of random mutagenesis. The strain was shown to possess an expanded endoplasmic reticulum, but the genetic factors responsible for this phenotype remain still unidentified. Recently, three new transcription factors were described in Neurospora crassa which were demonstrated to be involved in protein secretion. One of them, RES2, was involved in upregulation of secretion-related genes. The aim of our present study was therefore to analyze the role of RES2, on protein secretion in the T. reesei Rut-C30 strain. RESULT: Deletion of the res2 gene in Rut-C30 resulted in slightly slower growth on all substrates tested, and lower germination rate as well as lower protein secretion compared to the parental strain Rut-C30. Transcriptomic analysis of the Rut-C30 and the Δres2 mutant strain in secretion stress conditions showed remarkably few differences : 971 genes were differentially expressed (DE) in both strains while 192 genes out of 1163 (~ 16.5%) were DE in Rut-C30 only and 693 out of 1664 genes (~ 41.6%) displayed differential expression solely in Δres2. Notably, induction of protein secretion by cultivating on lactose and addition of secretion stress inducer DTT induced many genes of the secretion pathway similarly in both strains. Among the differentially expressed genes, those coding for amino acid biosynthesis genes, transporters and genes involved in lipid metabolism were found to be enriched specifically in the Δres2 strain upon exposure to lactose or DTT. Besides, redox homeostasis and DNA repair genes were specifically upregulated in the Δres2 strain, indicating an altered stress response. CONCLUSION: These results indicate that in the T. reesei Rut-C30 strain, RES2 does not act as a master regulator of the secretion pathway, but it contributes to a higher protein secretion by adjusting the expression of genes involved in different steps of protein synthesis and the secretion pathway.

Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus.

Introduction: Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, is used in industry to produce lignocellulolytic enzymes and heterologous proteins. However, the transcriptional network driving the expression of these proteins remains elusive. As a first step to systematically uncover this network, we investigated growth, protein secretion, and transcriptomic fingerprints of strains deficient in the cellulolytic transcriptional regulators Clr1, Clr2, and Clr4, respectively. Methods: The genes encoding Clr1, Clr2, and Clr4 were individually deleted using split marker or the CRISPR/Cas12a technology and the resulting strains as well as the parental strain were cultivated in bioreactors under chemostat conditions using glucose as the carbon source. During steady state conditions, cellulose was added instead of glucose to study the genetic and cellular responses in all four strains to the shift in carbon source availability. Results: Notably, the clr1 and clr2 deletion strains were unable to continue to grow on cellulose, demonstrating a key role of both regulators in cellulose catabolism. Their transcriptomic fingerprints uncovered not only a lack of cellulase gene expression but also reduced expression of genes predicted to encode hemicellulases, pectinases, and esterases. In contrast, the growth of the clr4 deletion strain was very similar compared to the parental strain. However, a much stronger expression of cellulases, hemicellulases, pectinases, and esterases was observed. Discussion: The data gained in this study suggest that both transcriptional regulators Clr1 and Clr2 activate the expression of genes predicted to encode cellulases as well as hemicellulases, pectinases, and esterases. They further suggest that Clr1 controls the basal expression of cellulases and initiates the main lignocellulolytic response to cellulose via induction of clr2 expression. In contrast, Clr4 seems to act as a repressor of the lignocellulolytic response presumably via controlling clr2 expression. Comparative transcriptomics in all four strains revealed potentially new regulators in carbohydrate catabolism and lignocellulolytic enzyme expression that define a candidate gene list for future analyses.

CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii.

BACKGROUND: The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed. RESULTS: A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), ß-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, ß-xylosidase, xylanase, ß-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3. CONCLUSIONS: Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Production of fermentable glucose from bioconversion of cellulose using efficient microbial cellulases produced from water hyacinth waste.

The economic production of cellulase enzymes for various industrial applications is one of the major research areas. A number of broad industrial applications, for example, in cellulosic biomass hydrolysis for simple sugars such as glucose and subsequent biofuel production, make these enzyme systems the third most demanding enzymes. Nevertheless, due to their production on commercial substrates, cellulases fall into the category of costly enzymes. Therefore, the goal of the present work is to evaluate the enhancement of cellulase production and its utilization in the enzymatic hydrolysis of biomass using low-cost cellulosic substrate, which is abundant and widely available. In this context, waste biomasses of water hyacinth (WH), including leaves and stems, have been used as feedstock to produce cellulases via solid-state fermentation (SSF) in the current study, which improves its production as well as activity. Furthermore, the impact of process parameters like temperature and pH has been investigated for improved cellulase production. At optimum concentration using 10 g of feedstock, 22 IU/gds of FP, 92 IU/gds of BGL, and 111 IU/gds of EG have been noticed in day 5 of SSF. Herein, 40 °C has been identified as the optimum temperature for cellulase production, whereas 50-55 °C has been recorded as the optimum reaction temperature for cellulase enzyme activity. Additionally, pH 5.5 has been identified as the optimum pH for cellulase enzyme production, whereas this enzyme was thermally stable (55 °C) at pH 5.0 up to 3.5 h. Further, the cellulosic biomass hydrolysis of WH leaves via an optimized crude enzyme has been performed, and this could release 24.34 g/L of glucose in 24 h of the reaction. The current findings may have potential for developing cellulases for mass-scale production using WH-based waste bioresources for numerous biorefinery applications.